Cell culture produced monoclonal antibodies (mAbs) have advantages over polyclonal antibodies derived from human or animal sera, such as reproducibility of production and enhanced safety profile. Although targeting multiple epitopes on cancer cells, viruses or toxins with multiple mAbs in combination has demonstrated superior therapeutic effects compared to mAb monotherapy, oligoclonal mAb therapy is not widely practiced partially because of the cost of separately producing and characterizing multiple mAbs for use in combination. In this proposal we will test a novel idea for producing an oligoclonal mixture of mAbs using a plant expression system. Plant viruses exhibit a phenomenon called superinfection exclusion, whereby a preexisting viral infection prevents a secondary infection with the same or a closely related virus. We propose to express multiple mAbs in a single plant simultaneously, exploiting the superinfection exclusion characteristic of plant virus expression vectors to ensure that each heavy chain forms a cognate pair with the appropriate light chain. The heavy and light chain sequences of three mAbs against botulinum neurotoxin serotype A (BoNT/A) will be cloned into our dual viral expression vector, and introduced into Agrobacterum tumefaciens. MAb transient expression will be initiated by infiltration of the Agrobacterium recombinants into benthamiana leaf tissue. Infiltration conditions will be optimized to obtain high-level expression, and to facilitate comparable expression levels of all three mAbs when co-expressed. We will demonstrate that three monoclonal antibodies can be expressed without mixing up heavy and light chains and that the combination can be manufactured reproducibly. The heterogeneity of the individual antibodies and the polyclonality of each batch will be monitored using cation exchange chromatography, taking advantage of the different isoelectric points of the three mAbs. We will then compare the in vivo potency of 1) three anti-BoNT/A mAbs prepared separately in plants and then combined with 2) the same mAbs produced from a single plant infiltration, using the mouse toxin neutralization assay. The percent survival will be determined and the potency (IU/mg) of each plantibody mixture will be calculated.